CN114853906A - Fusion protein of recombinant forest encephalitis virus, recombinant forest encephalitis virus bacteria-like particle vaccine and application of fusion protein and bacterial-like particle vaccine - Google Patents

Abstract

The invention discloses a fusion protein of a recombinant forest encephalitis virus, a recombinant forest encephalitis virus bacteria-like particle vaccine and application of the fusion protein and the recombinant forest encephalitis virus bacteria-like particle vaccine, and belongs to the technical field of vaccines. To provide a tick-borne encephalitis virus vaccine with better immune efficacy. The invention provides a fusion protein of a recombinant forest encephalitis virus, which is a forest encephalitis virus envelope protein E protein, a Linker is utilized to connect an anchor protein PA3 and an antigen protein in series to obtain the fusion protein, the fusion protein is combined with GEM particles, and the antigen protein is displayed on the surfaces of the GEM particles to prepare the recombinant forest encephalitis virus bacterial-like particle vaccine. The GEM particles have good antigen surface display properties, and can induce mice to generate specific immune response.

Description

Fusion protein of recombinant forest encephalitis virus, recombinant forest encephalitis virus bacteria-like particle vaccine and application of fusion protein and bacterial-like particle vaccine

Technical Field

The invention belongs to the technical field of vaccines, and particularly relates to a fusion protein of a recombinant tick-borne encephalitis virus, a recombinant tick-borne encephalitis virus bacterial-like particle vaccine and application of the fusion protein and the recombinant tick-borne encephalitis virus bacterial-like particle vaccine.

Background

Tick-borne encephalitis (TBE), a neurotropic disease caused by Tick-borne encephalitis virus, is ubiquitous in the continental europe. In recent years, the incidence and prevalence of the disease have increased. Can cause serious acute clinical course and long-term symptoms, causes mild or moderate febrile diseases in human, and is accompanied with sequelae such as lethal encephalitis, and the fatality rate is as high as 30%. At present, no specific treatment method for the forest encephalitis exists, but the immunization can effectively prevent the forest encephalitis.

Forest-borne encephalitis virus (TBEV) is a single-stranded positive-strand RNA virus of the flaviviridae genus, which is about 11kb in length, and comprises an open reading frame encoding three structural proteins (C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS 5). Wherein the E protein is envelope protein, contains neutralizing epitope, and plays an important role in immunoprophylaxis. In 2005, the international committee for virus nomenclature divided TBEVs into three subtypes according to genotype: the European subtype (Eu-TBEV), the Siberian subtype (Sib-TBEV) and the far east subtype (FE-TBEV), each of which may cause clinical disease of varying severity. In recent years, on the basis of the separation of the Begal subtype (Bkl-TBEV) and the Himalayan subtype (Him-TBEV), no corresponding cases are found, and the pathogenicity of the Himalayan subtype is further researched. Among them, FE-TBEV has the strongest toxicity, can cause serious nervous system diseases, has the fatality rate as high as 30 percent, and is easy to cause nervous system sequelae. Currently, tick-borne encephalitis has no specific curative medicine and can only be treated according to symptoms clinically, and although the currently approved inactivated vaccines show good safety and immunogenicity, multiple immunizations are needed, the cost is high, and incomplete inactivation can occur, so that a novel TBEV vaccine which is high in safety, low in cost and capable of stimulating the organism to generate high immunity is urgently needed in the market.

Traditional inactivated vaccines (these traditional vaccines are all inactivated vaccines) FSME-Immun and Encepur, the basic immunization program requires 3 doses, the vaccination program is 1, 2 doses separated by 1-3 months, 2, 3 doses separated by 5-12 months (Encepur requires 9-12 months), Encepur can be vaccinated at 0, 7, 21d, and then 4 months.

The traditional inactivated vaccines TBE-Moscow and Ence Vir are used for 2 times, and the Ence Vir is not recommended to be used for people of 3-17 years old, and may cause high fever and allergic reaction of less than or equal to 19 percent of recipients, and the safety is low.

Disclosure of Invention

The invention aims to provide a tick-borne encephalitis virus vaccine which is high in safety, low in cost and good in immune efficacy.

The invention provides a fusion protein of a recombinant forest encephalitis virus, which is a forest encephalitis virus envelope protein E protein, and a Linker is used for connecting a fluke protein PA3 and the forest encephalitis virus envelope protein E protein in series to obtain the fusion protein.

Further limited, the amino acid sequence of the Linker is shown as SEQ ID NO. 4.

Further limited, the amino acid sequence of the anchor hook protein PA3 is shown in SEQ ID NO. 6.

Further limited, the amino acid sequence of the forest encephalitis virus envelope protein E protein is shown in SEQ ID NO. 2.

Further limited, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 8.

The invention provides a gene encoding the fusion protein.

The invention provides an application of a forest encephalitis virus envelope protein E protein in preparation of a forest encephalitis virus vaccine.

Further limited, the amino acid sequence of the forest encephalitis virus envelope protein E protein is shown in SEQ ID NO. 2.

The invention provides a recombinant bacteria-like particle vaccine for forest encephalitis virus, which is prepared by displaying envelope protein E protein of the forest encephalitis virus on the surface of GEM particles.

The invention provides a preparation method of the fusion protein, which is characterized by comprising the following steps:

(1) connecting a gene encoding the anchor hook protein PA3 protein with a gene encoding the forest encephalitis virus envelope protein E protein through a gene encoding a Linker to obtain a recombinant gene;

(2) connecting the recombinant gene obtained in the step (1) with an expression vector pFastBac Dual to obtain a recombinant plasmid;

(3) transforming the recombinant plasmid obtained in the step (2) into a DH10Bac competent cell to obtain recombinant engineering bacteria, and extracting recombinant bacmid in the recombinant engineering bacteria;

(4) transfecting the recombinant bacmid obtained in the step (3) into Sf9 cells to obtain recombinant viruses, culturing the recombinant viruses to obtain cell suspension, wherein the fusion protein is in the cell suspension.

The invention provides application of the fusion protein or the recombinant forest encephalitis virus bacterial-like particle vaccine in preparation of medicines for treating or preventing diseases infected with forest encephalitis virus.

Has the advantages that: the application provides a encephalitis disease in forestA toxic GEM particle vaccine is prepared by connecting anchor hook protein PA3 and forest encephalitis virus envelope protein E in series by a Linker, expressing the E-Linker-PA3 by using an insect cell-baculovirus expression system, and displaying a recombinant antigen E-Linker-PA3 prepared by a Bac-to-Bac expression system on the surface of GEM particles, wherein the GEM particles have good antigen surface display property and can induce a mouse to generate specific immune response, and the immune memory can be generated after the mouse is immunized by intramuscular injection for 2 times of immunization, so that an organism can be stimulated to generate stronger cellular immunity and humoral immunity, and the mouse is induced to generate higher specific IgG antibody (maximum 1: 10) ^6.9 ) IgG levels 1 at 24 weeks after the last immunization: 10 ^6.0 。

Drawings

FIG. 1 is a schematic representation of the recombinant plasmid pFBD-E-Linker-PA3-E-Linker-PA 3.

FIG. 2 shows the result of PCR amplification of the E-Linker-PA3 gene; wherein, A: TBEV E and Linker-PA3 gene amplification results; b: E-Linker-PA3(PH) gene PCR amplification result; c: E-Linker-PA3(P10) gene PCR amplification result. 1: PCR amplification products of TBEV E gene; 2: PCR amplification products of Linker-PA3 gene; 3: PCR amplification products of E-Linker-PA3 (PH); 4: PCR amplification products of E-Linker-PA3 (P10).

FIG. 3 is an identification of recombinant plasmid pFBD-E-Linker-PA3-E-Linker-PA 3; wherein, A: a pFBD-E-Linker-PA3 double enzyme digestion identification result; b: and carrying out double-enzyme digestion identification on the pFBD-E-Linker-PA3-E-Linker-PA 3. 1: a pFBD-E-Linker-PA3 enzyme digestion product; 2: pFBD-E-Linker-PA3-E-Linker-PA3 enzyme digestion product.

FIG. 4 is a PCR identification of recombinant bacmid-E-Linker-PA 3; wherein, 1, rBacmid-E-Linker-PA3 PH end identification result; 2, rBacmid-E-Linker-PA 3P 10 end identification result.

FIG. 5 is the morphological change of Sf9 cells after infection with recombinant baculovirus; wherein, A: normal Sf9 cells; b: the recombinant baculovirus rBV-E-Linker-PA3 infected Sf9 cells.

FIG. 6 is the identification of recombinant baculovirus rBV-E-Linker-PA 3; wherein, A: indirect immunofluorescence identification (A-a: normal Sf9 cells; A-b: recombinant baculovirus rBV-E-Linker-PA3 infected Sf9 cells); b: western Blot identification (1: rBV-E-Linker-PA3 cell suspension; 2: rBV-E-Linker-PA3 culture supernatant; 3: rBV-E-Linker-PA3 cell pellet suspension).

FIG. 7 is an identification of TBEV BLPs; wherein, A: western Blot identification (1: GEM particle control; 2: TBEV BLPs); b: indirect immunofluorescence assay (B-a: GEM particles; B-B: TBEV BLPs); c: electron microscopy (C-a: GEM particles; C-b: TBEV BLPs).

FIG. 8 shows the detection of TBEV IgG antibodies in mouse serum.

FIG. 9 is a flow cytometric analysis of immune cells in the inguinal lymph node after priming; wherein, A: activation of mouse lymph node DC cells (A-a: CD11 c) ⁺ CD80 ⁺ The proportion of double positive cells; a-b CD11c ⁺ MHCⅠ ⁺ The proportion of double positive cells; a-c CD11c ⁺ MHCⅡ ⁺ Double positive cell proportion); b: recruitment and activation of B cells from mouse lymph nodes (B-a: CD 19) ⁺ CD40 ⁺ The proportion of double positive cells; B-B CD19 ⁺ CD69 ⁺ Double positive cell ratio).

FIG. 10 is an ELISpot assay for spleen cells IFN-. gamma.and IL-4; wherein, A: ELISpot detection of spleen cell IFN-gamma; b: ELISpot detection of splenocyte IL-4.

FIG. 11 is a spleen cell proliferation assay.

FIG. 12 is cytokine detection; wherein, A: th1 type cytokines; b: th2 type cytokine.

FIG. 13 is a flow cytometric analysis of mouse splenocytes after boosting; wherein, A: recruitment and activation of murine splenic T cells (A-a: CD 4) ⁺ CD69 ⁺ The proportion of double positive cells; a-b CD8 ⁺ CD69 ⁺ Double positive cell proportion); b: production of mouse spleen memory T cells (B-a: CD 4) ⁺ /CD44 ⁺ CD62L ⁺ The proportion of double positive cells; B-B CD8 ⁺ /CD44 ⁺ CD62L ⁺ Double positive cell ratio).

Detailed Description

pFastBac Dual is a commercially available carrier.

Example 1 construction and identification of recombinant baculovirus rBV-E-Linker-PA3

1. Primer design and Synthesis

Specific amplification primers were designed (table 1). An upstream primer E-PH-F (added with Not I enzyme cutting site) and a downstream primer E-R (added with 21bp Linker gene at the 3' end) are designed by referring to the envelope protein E gene of TBEV far east subtype strain WH2012 (GenBank: KJ 755186). WH2012-TBEV-E (without transmembrane domain), DNA, 1251bp, and the gene sequence is shown in SEQ ID NO. 1. WH2012-TBEV-E (without transmembrane domain), amino acid, 417aa, and the amino acid sequence is shown in SEQ ID NO. 2.

An upstream primer Linker-PA3-F (a C-terminal gene of 22bp TBEV envelope protein E is added to the 5' end) and a downstream primer Linker-PA3-R (HindIII enzyme cutting sites are added) are designed according to a Linker-PA3 gene stored in a laboratory. Linker, DNA, 24bp, gene sequence shown as SEQ ID NO.3, 4Linker, amino acid, 8aa, and amino acid sequence shown as SEQ ID NO. 4.

PA3 gene sequence, DNA, 672bp, as shown in SEQ ID NO. 5. PA3, amino acid 224aa, the amino acid sequence is shown in SEQ ID NO. 6.

The E gene, the Linker gene and the PA3 gene are connected to obtain E-Linker-PA3, DNA, 1947bp, and the sequence is shown as SEQ ID NO. 7. E-Linker-PA3, amino acid 649aa, and the sequence is shown in SEQ ID NO. 8.

The process for constructing the pFBD-E-Linker-PA3 vector comprises the following steps: the envelope protein E gene is connected with a Linker-PA3 gene and indirectly connected to a pFastBac Dual vector to obtain pFBD-E-Linker-PA 3.

An upstream primer E-P10-F (added with Sma I enzyme cutting sites) and a downstream primer Linker-PA3-P10-R (added with Nhe I enzyme cutting sites) are designed by taking a recombinant plasmid pFBD-E-Linker-PA3 constructed at the PH end as a template.

PH-terminal identifying primers PHF and M13R and P10-terminal identifying primers M13F and P10R are designed according to an insect cell-baculovirus expression vector pFastBac Dual.

TABLE 1 primer information

Note: the restriction sites are underlined.

The method for constructing the recombinant plasmid pFBD-E-Linker-PA3-E-Linker-PA3 comprises the following steps: E-Linker-PA3 and pFBD-E-Linker-PA3 are connected to obtain pFBD-E-Linker-PA3-E-Linker-PA3 recombinant plasmid.

A schematic representation of the pFBD-E-Linker-PA3-E-Linker-PA3 recombinant plasmid is shown in FIG. 1,

2. construction and identification of recombinant plasmid pFBD-E-Linker-PA3-E-Linker-PA3

(1) Amplification of target Gene E-Linker-PA3(PH)

The TBEV-E gene was amplified using the TBEV E gene as a template and E-PH-F and E-R as primers (FIG. 2A). A plasmid containing a Linker-PA3 gene is used as a template, Linker-PA3-F and Linker-PA3-R are used as primers to amplify the Linker-PA3 gene (figure 2A), the reaction conditions are shown in Table 2, and two target fragments are separated and recovered through 1% agarose gel electrophoresis. Amplified TBEV-E and Linker-PA3 genes are used as templates, E-PH-F and Linker-PA3-R are used as primers, the E-Linker-PA3(PH) gene is amplified by an overlap method, and the two target segments are connected in series. The amplified product was identical in size to the target fragment (FIG. 2B), and the E-Linker-PA3(PH) gene was successfully amplified.

TABLE 2 PCR reaction conditions

(2) Construction and identification of recombinant plasmid pFBD-E-Linker-PA3

The recovered E-Linker-PA3(PH) gene and pFastBac Dual vector are subjected to double enzyme digestion by NotI and Hind III, the enzyme-digested products are connected and transformed into Stellar competent cells, the Stellar competent cells are coated on an ampicillin-resistant solid LB plate, after inverted culture is carried out for 12h at 37 ℃, a monoclonal strain is selected to be shake-cultured in ampicillin-resistant liquid LB for 12h, plasmids are extracted and subjected to double enzyme digestion identification by NotI and Hind III (figure 3A), and the correct plasmids are identified for sequence determination and analysis, so that the sequence homology of the plasmids with the synthesized TBEV-E-PA3 gene is 100 percent, the recombinant plasmids containing the E-Linker-PA3 gene are successfully constructed, and the plasmids are named as pFBD-E-Linker-PA 3.

(3) Amplification of target Gene E-Linker-PA3(P10)

TBEV-E-PA3(P10) gene is amplified by taking the recombinant plasmid pFBD-E-Linker-PA3 as a template and E-P10-F and Linker-PA3-P10-R as primers (FIG. 2C). The amplified product is consistent with the size of the target gene fragment through agarose gel electrophoresis analysis, and the TBEV-E-PA3(P10) gene amplification is successful.

(4) Construction and identification of recombinant plasmid pFBD-E-Linker-PA3-E-Linker-PA3

The recovered E-Linker-PA3(P10) gene and recombinant plasmid pFBD-E-Linker-PA3 are subjected to double enzyme digestion by SmaI and Nhe I, the products after enzyme digestion are connected and transformed into Stellar competent cells, a monoclonal strain is selected, after the plasmid is extracted, double enzyme digestion identification is carried out by SmaI and Nhe I (figure 3B), the size of the enzyme digestion product is consistent with that of the E-Linker-PA3 gene, the recombinant plasmid containing the E-Linker-PA3 gene is successfully constructed, and the recombinant plasmid is named as pFBD-E-Linker-PA3-E-Linker-PA 3.

3. Preparation and identification of recombinant bacmid-E-Linker-PA3

The recombinant plasmid pFBD-E-Linker-PA3-E-Linker-PA3 was transformed into DH10Bac competent cells and plated on triple-resistant solid LB plates containing tetracycline (10. mu.g/mL), kanamycin sulfate (50. mu.g/mL), gentamicin (7. mu.g/mL), IPTG and X-Gal, and cultured upside down at 37 ℃ for 40 h. Picking white spots, shaking and culturing at 37 ℃ and 200rpm for 16h, and extracting bacmids for PCR identification. Identifying a target gene under a PH promoter by taking the bacmid as a template and PHF and M13R as primers; M13F and P10R were used as upstream and downstream primers to identify the target gene under the P10 promoter, and the PCR reaction is shown in Table 3. The results showed that the amplified product corresponded to the size of the fragment of interest (FIG. 4), indicating that the recombinant bacmid was successfully prepared and that the correctly identified bacmid was designated as rBacmid-E-Linker-PA 3.

TABLE 3 PCR reaction conditions

4. Rescue of recombinant baculovirus rBV-E-Linker-PA3

Inoculating Sf9 cells into a six-hole plate, culturing for 12h at 27 ℃, changing to a double-non Grace's culture medium when the cells grow to more than 80%, respectively diluting positive recombinant bacmid-E-Linker-PA3(4 mu g) and Cellffectin II Reagent (8 mu L) by using the double-non Grace's culture medium, slightly mixing uniformly, standing for 20min, uniformly adding into the 6-hole plate, standing and culturing for 5h at 27 ℃, changing to a Grace's complete culture medium, standing and culturing for 5 days at 27 ℃, and observing the cell state every day. Compared with normal adherent Sf9 cells, the adherent Sf9 cells transfected by the recombinant bacmid-E-Linker-PA3 have obvious cytopathic phenomena of expansion, rounding, large shedding and the like (figure 5). When 60% of cells are diseased on the 5 th day, collecting the supernatant as a first generation recombinant baculovirus named rBV-E-Linker-PA3, and continuing to pass through 3 generations blindly.

5. Identification of recombinant baculovirus rBV-E-Linker-PA3

(1) Indirect immunofluorescence assay

Sf9 cells with good growth state are uniformly paved into a six-hole plate, when the cells grow to 70%, recombinant baculovirus is inoculated, 10 mu L of recombinant baculovirus is inoculated in each hole, the culture medium is discarded after the culture is carried out for 48 hours at the temperature of 27 ℃. The cells were fixed with 70% ethanol for 30min, 100-fold diluted anti-TBEV E-DIII mouse serum (1% BSA) was used as the primary antibody, incubated at 37 ℃ for 1h, 200-fold diluted FITC-labeled goat anti-mouse IgG was used as the secondary antibody, and incubated at 37 ℃ for 1h in the dark. At the end of each run, the cells were washed 3 times with PBS. Cells were observed under an inverted fluorescence microscope. The results show (fig. 6A) that adherent Sf9 cells infected with recombinant baculovirus showed significant green fluorescence under fluorescent microscope compared to normal cell control, indicating that TBEV E protein was successfully expressed after recombinant baculovirus infected cells.

(2) Western Blot identification

Infecting Sf9 cells cultured in suspension by using recombinant baculovirus, culturing for 96 hours at the temperature of 27 ℃ at 120rpm, collecting the cells and culture supernatant by low-speed centrifugation, preparing samples by using cell suspension, culture supernatant and cell precipitate suspension, and carrying out Western Blot identification by using anti-TBEV E-DIII mouse serum diluted at the ratio of 1:300 as a primary antibody and HRP-labeled goat anti-mouse IgG diluted at the ratio of 1:20000 as a secondary antibody. The results show (FIG. 6B) that a specific protein band of about 70kD could be detected in recombinant baculovirus culture supernatant, consistent with the protein size predicted by the software. In conclusion, the recombinant baculovirus can successfully express the TBEV-E-PA3 protein after infecting Sf9 cells, and can secrete the protein to be expressed into a supernatant.

Example 2 preparation and characterization of TBEV BLPs

1. Preparation of TBEV BLPs

GEM particles were prepared from trichloroacetic acid and spin-bound to the TBEV-E-PA3 protein obtained in example 1 for 1h at room temperature. After anchoring and binding of PA3 to GEM particles, TBEV BLPs were prepared by centrifugation at 8000g at 4 ℃ for 10min, washing 5 times with sterile PBS, and suspending the resulting precipitate with PBS.

2. Western Blot identification

Western Blot identification was performed by taking GEM particles and TBEV BLPs as samples, using 1:300 diluted anti-TBEV E-DIII mouse serum as a primary antibody, and 1:20000 diluted HRP-labeled goat anti-mouse IgG as a secondary antibody. The results show (FIG. 7A) that a specific protein band appears at 70kD, indicating that the fusion protein TBEV-E-Linker-PA3 was successfully anchored to the GEM particles.

3. Indirect immunofluorescence assay

GEM particles and TBEV BLPs were sampled, blocked with 3% BSA at room temperature for 30min, and incubated at room temperature for 1h with 1:200 diluted TBEV E-DIII mouse serum as the primary antibody. Washed 3 times with PBS, and incubated for 1h at room temperature in the dark with FITC-labeled goat anti-mouse IgG diluted at a ratio of 1:200 as a secondary antibody. Then washed 3 times with PBS, resuspended in deionized water, precipitated, dropped onto a slide, and observed with a fluorescence microscope, showing that (FIG. 7B), the TBEV BLPs were visualized as bright green particles under the fluorescence microscope, indicating that the fusion protein TBEV-E-Linker-PA3 was successfully anchored to the GEM particles.

4. Transmission electron microscopy of TBEV BLPs

Fresh GEM particles and TBEV BLPs are taken to be 1U and 8000g respectively, centrifuged for 2min, the supernatant is carefully discarded, the sediment is fixed by a stationary liquid, embedded and sliced, and the morphological structure of the particles is observed under a transmission electron microscope. As a result, it was found (fig. 7C) that GEM particles were intact in structure, contained less content, and had clean surfaces; the GEM particles bound with the fusion protein TBEV-E-PA3 have a large amount of floccules on the surface. In conclusion, the fusion protein TBEV-E-Linker-PA3 is anchored on the surface of GEM particles, and the TBEV BLPs are successfully prepared.

Example 3 evaluation of immune Effect of TBEV BLPs

1. Immunization of mice

To evaluate the immune effect of TBEV BLPs, 27 healthy female BALB/C mice, 16-18g, were randomly divided into 3 groups, and the experimental group mice were treated with purified TBEV BLPs as antigen supplemented with the complex adjuvant Poly (I: C) & Montanide ISA 201 VG. According to the antigen: poly (I: C): montanide ISA 201VG 35: 10: 55, mixing the antigen with Poly (I: C), emulsifying with Montanide ISA 201VG (preheated at 31 ℃), shaking for 10min on a vortex oscillator, and standing for 1h at 21 ℃ after complete emulsification. Each mouse was injected intramuscularly with 100 μ L of immunogen, the grouping protocol is shown in table 4 below:

TABLE 4 BALB/c mouse immunization and grouping protocol

2. Detection of mouse serum TBEV IgG antibody

The serum IgG antibody levels of mice at 1 (1 week after first immunization), 3, 4 (1 week after second immunization), 6, 8, 10, 12, 16, 20 and 24 weeks after immunization with TBEV BLPs were determined by indirect ELISA using PBS group mouse serum as a negative control. The method comprises the following steps: the purified recombinant protein TBEV E-DIII expressed in pronucleus with the final concentration of 1 mug/mL is used as a coating antigen and coated overnight at 4 ℃. 5% of skimmed milk powder is used as a sealing liquid; from 1: diluting the serum of the mouse to be detected as a primary antibody at a ratio of 2 times at the beginning of 100; mixing the raw materials in a ratio of 1: a 40000-fold dilution of HRP-labeled goat anti-mouse IgG antibody was used as the secondary antibody. Developing with TMB for 3 min, and applying 0.5M H ₂ SO ₄ The color development was terminated. Reading OD value of each hole at the wavelength of 450nm of an enzyme labeling instrument, and determining the OD of the serum to be detected ₄₅₀ Negative serum OD ₄₅₀ The highest serum dilution at > 2.1 was taken as the ELISA antibody titer.

The results show (fig. 8): TBEV-specific IgG antibodies (1: 10) can be detected in mouse serum 1 week after initial immunization ^1.7 ) (ii) a Post-secondary immunization mouse serum IThe level of the gG antibody is obviously improved and can reach 1:10 at most ^6.9 (average value 1: 10) ^6.3 ) (ii) a The level of TBEV IgG antibody in the serum of the mouse slowly decreases at the beginning of 8 weeks and can still be maintained at 1:10 in 5 months after the second immunization ⁶ 。

3. Flow cytometry analysis of immune cells in inguinal lymph nodes

In 1 week after the first immunization, 3 mice in TBEV BLPs, Adjuvant and PBS groups are randomly selected, inguinal lymph nodes of the mice are respectively taken to prepare lymphocyte suspensions, cells are stained, and fluorescent signals are detected on a flow cytometry.

(1) Activation assay for mouse lymph node DC cells

Mouse inguinal lymph node cell line CD80 ⁺ 、MHCⅠ ⁺ 、MHCⅡ ⁺ And CD11c ⁺ Detection of CD11c after fluorescent antibody staining ⁺ CD80 ⁺ 、CD11c ⁺ MHCⅠ ⁺ And CD11c ⁺ MHCⅡ ⁺ Double positive cell ratio. The results show that: immune group mouse CD11c ⁺ CD80 ⁺ The proportion of double positive cells is significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.001) (FIG. 9A-a); immune group mouse CD11c ⁺ MHCⅠ ⁺ The proportion of double positive cells was significantly higher than that of the PBS control group (P < 0.001) and the Adjuvant control group (P < 0.01) (FIG. 9A-b); immune group mouse CD11c ⁺ MHCⅡ ⁺ The proportion of double positive cells was significantly higher than that of PBS control group (P < 0.0001) and Adjuvant control group (P < 0.001) (FIGS. 9A-c). In summary, mice first immunized with TBEV BLPs promoted activation of lymph node DC cells (. P < 0.05,. P < 0.01,. P < 0.001,. P < 0.0001).

(2) Recruitment and activation of B cells from mouse lymph nodes

Mouse inguinal lymph node cells, CD19 ⁺ 、CD40 ⁺ And CD69 ⁺ Detection of CD19 after fluorescent antibody staining ⁺ CD40 ⁺ And CD19 ⁺ CD69 ⁺ Double positive cell ratio (fig. 9B). The results show that: immunization group mouse CD19 ⁺ CD40 ⁺ The proportion of double positive cells is significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.001) (FIG. 9B-a); immunization group mouse CD19 ⁺ CD69 ⁺ Double positive thinThe cell ratio was significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.0001) (FIG. 9B-B). Taken together, there was significant recruitment and/or activation of B cells in lymph nodes following the first immunization of mice with TBEV BLPs.

4. Spleen cell activation assay

Separating mouse spleen cell, adjusting the concentration of spleen cell to 2.5 × 10 ⁶ one/mL for use.

(1) ELISpot assay

Detection was performed according to the instructions of the mouse IFN-. gamma.and IL-4ELISpot detection kit, and the steps were as follows: adding 5X 10 of the mixture into each hole ⁵ The spleen cells were simultaneously stimulated with TBEV E-DIII at a final concentration of 10. mu.g/mL, and ELISpot plates were coated with tinfoil paper at 37 ℃ with 5% CO ₂ Culturing for 38h in a cell culture incubator, and using biotin-labeled detection antibody (BVD6-24G2-biotin) diluted at a ratio of 1:1000 as a primary antibody; 1:1000 diluted Streptavidin-HRP is used as a secondary antibody, a TMB color development solution is added after room temperature incubation, the color development is carried out in a dark place until clear spots appear, and the ELISpot plate is washed by tap water to stop the color development. And naturally air-drying the ELISpot plate in a dark place, performing full-automatic Spot image acquisition and counting by using an AID enzyme-linked Spot image automatic analyzer, and storing the number of Spot-Forming Cells (SFCs) and Spot images in each hole. The number of cell spots of the immune group and the control group in the presence of the stimulant is plotted, and the result shows that the number of the cell spots secreting IFN-gamma in spleen cells of mice in the immune group is remarkably higher than that of a PBS control group (P is less than 0.01) and that of an Adjuvant control group (P is less than 0.05 and 10A) after the mice are stimulated by TBEV specific antigen, and the number of the cell spots secreting IL-4 is remarkably higher than that of the PBS control group and that of the Adjuvant control group (P is less than 0.0001 and 10B). IFN-gamma and IL-4 are representative cytokines of Th1 type cell immunity and Th2 type humoral immunity respectively, after mice are immunized by TBEV BLPs, bodies can be induced to secrete Th1 type and Th2 type cytokines, and the difference of Th2 type cytokines is more obvious. In conclusion, after the TBEV BLPs are immunized, the body can be induced to generate Th1 type cellular immune response and Th2 type humoral immune response.

(2) Splenic lymphocyte proliferation assay

The proliferation capacity of splenic lymphocytes in vitro was examined by the CCK-8 method. Adjusting cell density to 2.5X 10 ⁶ Per mL and at 10mu.L/well was plated in 96-well cell culture plates, and splenic lymphocytes were stimulated with the recombinant protein TBEV E-DIII (final concentration 10. mu.g/mL), with no stimulation of wells. At 37 ℃ 5% CO ₂ The cells were cultured in an incubator for 44 hours, 10. mu.L of CCK-8 solution was added to each well, and the incubation in the incubator was continued for 4 hours. Detection of OD on microplate reader _450nm And (4) calculating the proliferation index. The results show (FIG. 11), the splenic lymphocyte proliferation index of the mice in the immunized group is significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.0001). In conclusion, after TBEV BLPs are immunized, the splenic lymphocyte proliferation of mice is obvious under the stimulation of specific antigens.

(3) Cytokine detection

And (3) detecting the secretion of Th1 and Th2 cytokines IFN-gamma, IL-12p70, TNF-alpha, IL-2, KC/GRO, IL-4, IL-5, IL-6, IL-10 and IL-1 beta in spleen lymphocytes under the action of TBEV specific stimulators by using an MSD detection technology. Adjusting the cell concentration to 2.5X 10 ⁶ one/mL, and at 5X 10 ⁵ One well was inoculated into a U-shaped 96-well plate and splenic lymphocytes were stimulated with the recombinant protein TBEV E-DIII (final concentration 10. mu.g/mL). At 37 ℃ 5% CO ₂ The cell culture box is used for culturing for 48 hours. Centrifuging at 4 deg.C in a dark place at 2500rpm for 10min, collecting 50 μ L of supernatant, storing in dry ice, and testing the content of each cytokine in the sample. The results show (fig. 12): after splenocytes are stimulated by TBEV specific stimulators, Th1 type cell factor TNF-alpha in splenocyte culture solution of mice in an immune group is obviously higher than that of a PBS control group (P is less than 0.01) and an Adjuvant control group (P is less than 0.0001); the Th2 type cytokines IL-5 and IL-6 in the splenocyte culture solution of the mice in the immune group are obviously higher than those in the Adjuvant control group (P < 0.05), and IL-10 and IL-1 beta are obviously higher than those in the PBS control group and the Adjuvant control group (P < 0.0001). In conclusion, after the mice are immunized with TBEV BLPs, Th1 type cellular immunity and Th2 type humoral immunity can be generated, and the Th2 type humoral immunity is taken as the main part, which is consistent with the ELISpot detection result of spleen cells IFN-gamma and IL-4.

(4) Spleen lymphocyte assay

Collecting the spleen lymphocytes after the centrifugation in the previous step, adding a fluorescent antibody premix, and detecting fluorescent signals of the lymphocytes by using a flow cytometer.

1) Recruitment and/or activation of mouse spleen T cells

Spleen lymphocytes are identified by CD4 ⁺ 、CD8 ⁺ And CD69 ⁺ Staining with fluorescent antibody, detecting CD4 ⁺ CD69 ⁺ And CD8 ⁺ CD69 ⁺ Double positive cell ratio. The results show that: immunization group mouse CD4 ⁺ CD69 ⁺ The proportion of double positive cells was not significantly different from that of both control groups (FIG. 13A-a)); immunization group mouse CD8 ⁺ CD69 ⁺ The proportion of double positive cells was significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.01) (FIGS. 13A-b). Taken together, splenic T cells recruited and/or activated upon immunization with TBEV BLPs under specific antigen stimulation.

2) Production of mouse spleen memory T cells

Spleen lymphocytes are identified by CD4 ⁺ 、CD8 ⁺ 、CD44 ⁺ And CD62L ⁺ Staining with fluorescent antibody, detecting CD4 ⁺ And CD8 ⁺ CD44 in Positive cells ⁺ CD62L ⁺ Double positive cell ratio. The results show that: immunization group mouse CD4 ⁺ /CD44 ⁺ CD62L ⁺ The proportion of double positive cells is significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.0001) (FIG. 13B-a); immunization group mouse CD8 ⁺ /CD44 ⁺ CD62L ⁺ The cell ratio was significantly higher than that of the PBS control group and the Adjuvant control group (P < 0.0001) (FIG. 13B-B). Taken together, spleen of mice after immunization with TBEV BLPs produced CD4 ⁺ And CD8 ⁺ Central memory T cells.

5. Safety tests:

BALB/c mice immunized for 10 weeks to 6 weeks, and mice immunized for 2 times have no side effect, and the safety experiment grouping scheme is shown in Table 5.

TABLE 5 grouping scheme for safety experiments

Comparative example 1.

TBEV BLPs: TBEV BLPs obtained in example 2 were produced by 2 immunizations using intramuscular injection of immunized miceHas effects in stimulating immune memory, stimulating cell immunity and humoral immunity, and inducing mouse to produce high specific IgG antibody (maximum 1: 10) ^6.9 IgG levels 1, 24 weeks after the last immunization: 10 ^6.0 )

Prokaryotic expression of TBEV E protein, purification by nickel column, intraperitoneal injection of immunized mice (500. mu.L/mouse) with aluminum adjuvant, IgG antibody titer: a maximum of 1: 6400, IgG levels had dropped to 1 at 7 weeks after the last immunization: 400.

SEQUENCE LISTING

<110> university of Jilin

<120> fusion protein of recombinant tick-borne encephalitis virus, recombinant tick-borne encephalitis virus bacterial-like particle vaccine and vaccine thereof

Applications of the same

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 1251

<212> DNA

<213> Artificial Synthesis

<400> 1

ggaggaaatg aaggctcaat catgtggctc gcgagcttgg cagttgtcat agcttgtgca 60

ggagccagcc gttgcacaca cctcgaaaac cgcgacttcg tgaccggcac acaaggaact 120

acacgcgtca ctctcgtcct ggagttggga ggttgcgtga ccatcaccgc tgagggcaag 180

ccaagcatgg acgtgtggct ggactccatc taccaggaga accccgctaa gacacgtgaa 240

tactgcctcc acgccaagct cagcgacaca aaggttgccg ctcgctgtcc tacaatgggc 300

ccagccaccc tggctgagga gcaccagagc ggtaccgtct gtaagcgcga ccagagcgac 360

cgcggttggg gcaatcactg cggcctcttc ggcaagggta gcatcgtgac ctgtgtgaag 420

gccagctgcg gagccaagaa gaaggccact ggccacgtgt acgacgctaa caagatcgtg 480

tacaccgtga aggtggagcc tcacaccggt gactacgtgg ctgctaacga gacccacagc 540

ggacgtaaga ccgccagctt caccgtcagc tccgaaaaga ctatcctcac tatgggcgac 600

tacggcgacg tgagcctgtt gtgtcgtgtt gccagcggcg tggacctcgc ccagacagtg 660

atcctggaac tggataagac atccgagcac ctgcccaccg cttggcaggt gcaccgtgac 720

tggttcaacg acctcgctct cccctggaag cacgaaggtg ctcagaactg gaacaacgct 780

gagcgtctgg tggagttcgg tgcccctcac gctgtgaaga tggacgttta caacttgggt 840

gaccagaccg gtgttctgct gaagtccctc gccggtgtgc ccgtcgctca cattgacggc 900

actaagtacc acctgaaaag cggccacgtg acttgtgagg ttggactgga gaagctgaag 960

atgaagggac tgacctacac aatgtgtgac aagactaagt tcacctggaa gcgtatccct 1020

acagactccg gacacgacac tgtcgtgatg gaggtggcct tcagcggtac taagccttgt 1080

cgtatcccag tgcgcgctgt ggctcacggc tcccctgacg tgaacgtggc tatgctgatc 1140

acccctaacc ctactatcga aaccaacggt ggcggcttca tcgagatgca gctgccaccc 1200

ggtgacaaca tcatctacgt tggtgaactg agccaccaat ggttccagaa g 1251

<210> 2

<211> 417

<212> PRT

<213> Artificial Synthesis

<400> 2

Gly Gly Asn Glu Gly Ser Ile Met Trp Leu Ala Ser Leu Ala Val Val

1 5 10 15

Ile Ala Cys Ala Gly Ala Ser Arg Cys Thr His Leu Glu Asn Arg Asp

20 25 30

Phe Val Thr Gly Thr Gln Gly Thr Thr Arg Val Thr Leu Val Leu Glu

35 40 45

Leu Gly Gly Cys Val Thr Ile Thr Ala Glu Gly Lys Pro Ser Met Asp

50 55 60

Val Trp Leu Asp Ser Ile Tyr Gln Glu Asn Pro Ala Lys Thr Arg Glu

65 70 75 80

Tyr Cys Leu His Ala Lys Leu Ser Asp Thr Lys Val Ala Ala Arg Cys

85 90 95

Pro Thr Met Gly Pro Ala Thr Leu Ala Glu Glu His Gln Ser Gly Thr

100 105 110

Val Cys Lys Arg Asp Gln Ser Asp Arg Gly Trp Gly Asn His Cys Gly

115 120 125

Leu Phe Gly Lys Gly Ser Ile Val Thr Cys Val Lys Ala Ser Cys Gly

130 135 140

Ala Lys Lys Lys Ala Thr Gly His Val Tyr Asp Ala Asn Lys Ile Val

145 150 155 160

Tyr Thr Val Lys Val Glu Pro His Thr Gly Asp Tyr Val Ala Ala Asn

165 170 175

Glu Thr His Ser Gly Arg Lys Thr Ala Ser Phe Thr Val Ser Ser Glu

180 185 190

Lys Thr Ile Leu Thr Met Gly Asp Tyr Gly Asp Val Ser Leu Leu Cys

195 200 205

Arg Val Ala Ser Gly Val Asp Leu Ala Gln Thr Val Ile Leu Glu Leu

210 215 220

Asp Lys Thr Ser Glu His Leu Pro Thr Ala Trp Gln Val His Arg Asp

225 230 235 240

Trp Phe Asn Asp Leu Ala Leu Pro Trp Lys His Glu Gly Ala Gln Asn

245 250 255

Trp Asn Asn Ala Glu Arg Leu Val Glu Phe Gly Ala Pro His Ala Val

260 265 270

Lys Met Asp Val Tyr Asn Leu Gly Asp Gln Thr Gly Val Leu Leu Lys

275 280 285

Ser Leu Ala Gly Val Pro Val Ala His Ile Asp Gly Thr Lys Tyr His

290 295 300

Leu Lys Ser Gly His Val Thr Cys Glu Val Gly Leu Glu Lys Leu Lys

305 310 315 320

Met Lys Gly Leu Thr Tyr Thr Met Cys Asp Lys Thr Lys Phe Thr Trp

325 330 335

Lys Arg Ile Pro Thr Asp Ser Gly His Asp Thr Val Val Met Glu Val

340 345 350

Ala Phe Ser Gly Thr Lys Pro Cys Arg Ile Pro Val Arg Ala Val Ala

355 360 365

His Gly Ser Pro Asp Val Asn Val Ala Met Leu Ile Thr Pro Asn Pro

370 375 380

Thr Ile Glu Thr Asn Gly Gly Gly Phe Ile Glu Met Gln Leu Pro Pro

385 390 395 400

Gly Asp Asn Ile Ile Tyr Val Gly Glu Leu Ser His Gln Trp Phe Gln

405 410 415

Lys

<210> 3

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 3

ggtggttctg gtggtggttc tggt 24

<210> 4

<211> 8

<212> PRT

<213> Artificial Synthesis

<400> 4

Gly Gly Ser Gly Gly Gly Ser Gly

1 5

<210> 5

<211> 672

<212> DNA

<213> Artificial Synthesis

<400> 5

gatggtgctt cttcagctgg taacaccaac tctggtggtt ccactacaac tatcactaac 60

aacaactctg gtactaactc ctcttctacc acctacaccg tgaagtctgg cgacactctg 120

tggggtatct cccaacgtta cggtatctct gttgctcaaa tccaatcagc taacaacctg 180

aagtctacta tcatctacat cggtcagaag ctggtgctga ccggttctgc ttcttctaca 240

aactctggag gttccaacaa ctccgcttcc actaccccta ccacttccgt gactcctgct 300

aagcctacct ctcagactac cgttaaggtg aagtcaggtg acactctgtg ggctttgtct 360

gtgaagtaca agacctccat cgctcagctg aagagctgga accacctgtc ctctgacact 420

atctacatcg gtcagaacct gatcgtgtcc cagtctgctg ctgcttccaa cccttcaaca 480

ggttctggtt ctaccgctac taacaactcc aactctactt cctcaaactc taacgcttca 540

atccacaagg tggtgaaggg tgacaccttg tggggtctgt cacagaagtc aggttccccc 600

atcgcttcta tcaaggcttg gaaccacctg tcttctgaca ccatcctgat cggtcaatac 660

ctgcgtatca ag 672

<210> 6

<211> 224

<212> PRT

<213> Artificial Synthesis

<400> 6

Asp Gly Ala Ser Ser Ala Gly Asn Thr Asn Ser Gly Gly Ser Thr Thr

1 5 10 15

Thr Ile Thr Asn Asn Asn Ser Gly Thr Asn Ser Ser Ser Thr Thr Tyr

20 25 30

Thr Val Lys Ser Gly Asp Thr Leu Trp Gly Ile Ser Gln Arg Tyr Gly

35 40 45

Ile Ser Val Ala Gln Ile Gln Ser Ala Asn Asn Leu Lys Ser Thr Ile

50 55 60

Ile Tyr Ile Gly Gln Lys Leu Val Leu Thr Gly Ser Ala Ser Ser Thr

65 70 75 80

Asn Ser Gly Gly Ser Asn Asn Ser Ala Ser Thr Thr Pro Thr Thr Ser

85 90 95

Val Thr Pro Ala Lys Pro Thr Ser Gln Thr Thr Val Lys Val Lys Ser

100 105 110

Gly Asp Thr Leu Trp Ala Leu Ser Val Lys Tyr Lys Thr Ser Ile Ala

115 120 125

Gln Leu Lys Ser Trp Asn His Leu Ser Ser Asp Thr Ile Tyr Ile Gly

130 135 140

Gln Asn Leu Ile Val Ser Gln Ser Ala Ala Ala Ser Asn Pro Ser Thr

145 150 155 160

Gly Ser Gly Ser Thr Ala Thr Asn Asn Ser Asn Ser Thr Ser Ser Asn

165 170 175

Ser Asn Ala Ser Ile His Lys Val Val Lys Gly Asp Thr Leu Trp Gly

180 185 190

Leu Ser Gln Lys Ser Gly Ser Pro Ile Ala Ser Ile Lys Ala Trp Asn

195 200 205

His Leu Ser Ser Asp Thr Ile Leu Ile Gly Gln Tyr Leu Arg Ile Lys

210 215 220

<210> 7

<211> 1947

<212> DNA

<213> Artificial Synthesis

<400> 7

ggaggaaatg aaggctcaat catgtggctc gcgagcttgg cagttgtcat agcttgtgca 60

ggagccagcc gttgcacaca cctcgaaaac cgcgacttcg tgaccggcac acaaggaact 120

acacgcgtca ctctcgtcct ggagttggga ggttgcgtga ccatcaccgc tgagggcaag 180

ccaagcatgg acgtgtggct ggactccatc taccaggaga accccgctaa gacacgtgaa 240

tactgcctcc acgccaagct cagcgacaca aaggttgccg ctcgctgtcc tacaatgggc 300

ccagccaccc tggctgagga gcaccagagc ggtaccgtct gtaagcgcga ccagagcgac 360

cgcggttggg gcaatcactg cggcctcttc ggcaagggta gcatcgtgac ctgtgtgaag 420

gccagctgcg gagccaagaa gaaggccact ggccacgtgt acgacgctaa caagatcgtg 480

tacaccgtga aggtggagcc tcacaccggt gactacgtgg ctgctaacga gacccacagc 540

ggacgtaaga ccgccagctt caccgtcagc tccgaaaaga ctatcctcac tatgggcgac 600

tacggcgacg tgagcctgtt gtgtcgtgtt gccagcggcg tggacctcgc ccagacagtg 660

atcctggaac tggataagac atccgagcac ctgcccaccg cttggcaggt gcaccgtgac 720

tggttcaacg acctcgctct cccctggaag cacgaaggtg ctcagaactg gaacaacgct 780

gagcgtctgg tggagttcgg tgcccctcac gctgtgaaga tggacgttta caacttgggt 840

gaccagaccg gtgttctgct gaagtccctc gccggtgtgc ccgtcgctca cattgacggc 900

actaagtacc acctgaaaag cggccacgtg acttgtgagg ttggactgga gaagctgaag 960

atgaagggac tgacctacac aatgtgtgac aagactaagt tcacctggaa gcgtatccct 1020

acagactccg gacacgacac tgtcgtgatg gaggtggcct tcagcggtac taagccttgt 1080

cgtatcccag tgcgcgctgt ggctcacggc tcccctgacg tgaacgtggc tatgctgatc 1140

acccctaacc ctactatcga aaccaacggt ggcggcttca tcgagatgca gctgccaccc 1200

ggtgacaaca tcatctacgt tggtgaactg agccaccaat ggttccagaa gggtggttct 1260

ggtggtggtt ctggtgatgg tgcttcttca gctggtaaca ccaactctgg tggttccact 1320

acaactatca ctaacaacaa ctctggtact aactcctctt ctaccaccta caccgtgaag 1380

tctggcgaca ctctgtgggg tatctcccaa cgttacggta tctctgttgc tcaaatccaa 1440

tcagctaaca acctgaagtc tactatcatc tacatcggtc agaagctggt gctgaccggt 1500

tctgcttctt ctacaaactc tggaggttcc aacaactccg cttccactac ccctaccact 1560

tccgtgactc ctgctaagcc tacctctcag actaccgtta aggtgaagtc aggtgacact 1620

ctgtgggctt tgtctgtgaa gtacaagacc tccatcgctc agctgaagag ctggaaccac 1680

ctgtcctctg acactatcta catcggtcag aacctgatcg tgtcccagtc tgctgctgct 1740

tccaaccctt caacaggttc tggttctacc gctactaaca actccaactc tacttcctca 1800

aactctaacg cttcaatcca caaggtggtg aagggtgaca ccttgtgggg tctgtcacag 1860

aagtcaggtt cccccatcgc ttctatcaag gcttggaacc acctgtcttc tgacaccatc 1920

ctgatcggtc aatacctgcg tatcaag 1947

<210> 8

<211> 649

<212> PRT

<213> Artificial Synthesis

<400> 8

Gly Gly Asn Glu Gly Ser Ile Met Trp Leu Ala Ser Leu Ala Val Val

1 5 10 15

Ile Ala Cys Ala Gly Ala Ser Arg Cys Thr His Leu Glu Asn Arg Asp

20 25 30

Phe Val Thr Gly Thr Gln Gly Thr Thr Arg Val Thr Leu Val Leu Glu

35 40 45

Leu Gly Gly Cys Val Thr Ile Thr Ala Glu Gly Lys Pro Ser Met Asp

50 55 60

Val Trp Leu Asp Ser Ile Tyr Gln Glu Asn Pro Ala Lys Thr Arg Glu

65 70 75 80

Tyr Cys Leu His Ala Lys Leu Ser Asp Thr Lys Val Ala Ala Arg Cys

85 90 95

Pro Thr Met Gly Pro Ala Thr Leu Ala Glu Glu His Gln Ser Gly Thr

100 105 110

Val Cys Lys Arg Asp Gln Ser Asp Arg Gly Trp Gly Asn His Cys Gly

115 120 125

Leu Phe Gly Lys Gly Ser Ile Val Thr Cys Val Lys Ala Ser Cys Gly

130 135 140

Ala Lys Lys Lys Ala Thr Gly His Val Tyr Asp Ala Asn Lys Ile Val

145 150 155 160

Tyr Thr Val Lys Val Glu Pro His Thr Gly Asp Tyr Val Ala Ala Asn

165 170 175

Glu Thr His Ser Gly Arg Lys Thr Ala Ser Phe Thr Val Ser Ser Glu

180 185 190

Lys Thr Ile Leu Thr Met Gly Asp Tyr Gly Asp Val Ser Leu Leu Cys

195 200 205

Arg Val Ala Ser Gly Val Asp Leu Ala Gln Thr Val Ile Leu Glu Leu

210 215 220

Asp Lys Thr Ser Glu His Leu Pro Thr Ala Trp Gln Val His Arg Asp

225 230 235 240

Trp Phe Asn Asp Leu Ala Leu Pro Trp Lys His Glu Gly Ala Gln Asn

245 250 255

Trp Asn Asn Ala Glu Arg Leu Val Glu Phe Gly Ala Pro His Ala Val

260 265 270

Lys Met Asp Val Tyr Asn Leu Gly Asp Gln Thr Gly Val Leu Leu Lys

275 280 285

Ser Leu Ala Gly Val Pro Val Ala His Ile Asp Gly Thr Lys Tyr His

290 295 300

Leu Lys Ser Gly His Val Thr Cys Glu Val Gly Leu Glu Lys Leu Lys

305 310 315 320

Met Lys Gly Leu Thr Tyr Thr Met Cys Asp Lys Thr Lys Phe Thr Trp

325 330 335

Lys Arg Ile Pro Thr Asp Ser Gly His Asp Thr Val Val Met Glu Val

340 345 350

Ala Phe Ser Gly Thr Lys Pro Cys Arg Ile Pro Val Arg Ala Val Ala

355 360 365

His Gly Ser Pro Asp Val Asn Val Ala Met Leu Ile Thr Pro Asn Pro

370 375 380

Thr Ile Glu Thr Asn Gly Gly Gly Phe Ile Glu Met Gln Leu Pro Pro

385 390 395 400

Gly Asp Asn Ile Ile Tyr Val Gly Glu Leu Ser His Gln Trp Phe Gln

405 410 415

Lys Gly Gly Ser Gly Gly Gly Ser Gly Asp Gly Ala Ser Ser Ala Gly

420 425 430

Asn Thr Asn Ser Gly Gly Ser Thr Thr Thr Ile Thr Asn Asn Asn Ser

435 440 445

Gly Thr Asn Ser Ser Ser Thr Thr Tyr Thr Val Lys Ser Gly Asp Thr

450 455 460

Leu Trp Gly Ile Ser Gln Arg Tyr Gly Ile Ser Val Ala Gln Ile Gln

465 470 475 480

Ser Ala Asn Asn Leu Lys Ser Thr Ile Ile Tyr Ile Gly Gln Lys Leu

485 490 495

Val Leu Thr Gly Ser Ala Ser Ser Thr Asn Ser Gly Gly Ser Asn Asn

500 505 510

Ser Ala Ser Thr Thr Pro Thr Thr Ser Val Thr Pro Ala Lys Pro Thr

515 520 525

Ser Gln Thr Thr Val Lys Val Lys Ser Gly Asp Thr Leu Trp Ala Leu

530 535 540

Ser Val Lys Tyr Lys Thr Ser Ile Ala Gln Leu Lys Ser Trp Asn His

545 550 555 560

Leu Ser Ser Asp Thr Ile Tyr Ile Gly Gln Asn Leu Ile Val Ser Gln

565 570 575

Ser Ala Ala Ala Ser Asn Pro Ser Thr Gly Ser Gly Ser Thr Ala Thr

580 585 590

Asn Asn Ser Asn Ser Thr Ser Ser Asn Ser Asn Ala Ser Ile His Lys

595 600 605

Val Val Lys Gly Asp Thr Leu Trp Gly Leu Ser Gln Lys Ser Gly Ser

610 615 620

Pro Ile Ala Ser Ile Lys Ala Trp Asn His Leu Ser Ser Asp Thr Ile

625 630 635 640

Leu Ile Gly Gln Tyr Leu Arg Ile Lys

645

<210> 9

<211> 59

<212> DNA

<213> Artificial Synthesis

<400> 9

taagcggccg catgggagga aatgaaggct caatcatgtg gctcgcgagc ttggcagtt 59

<210> 10

<211> 44

<212> DNA

<213> Artificial Synthesis

<400> 10

agaaccacca ccagaaccac cggcgccgac acccagggtc atag 44

<210> 11

<211> 43

<212> DNA

<213> Artificial Synthesis

<400> 11

tatgaccctg ggtgtcggcg ccggtggttc tggtggtggt tct 43

<210> 12

<211> 33

<212> DNA

<213> Artificial Synthesis

<400> 12

tataagcttt tacttgatac gcaggtattg acc 33

<210> 13

<211> 46

<212> DNA

<213> Artificial Synthesis

<400> 13

acgaagactt gatcacccgg gatgggagga aatgaaggct caatca 46

<210> 14

<211> 45

<212> DNA

<213> Artificial Synthesis

<400> 14

tgctatgcat cagctgctag cttacttgat acgcaggtat tgacc 45

<210> 15

<211> 23

<212> DNA

<213> Artificial Synthesis

<400> 15

cccagtcacg acgttgtaaa acg 23

<210> 16

<211> 19

<212> DNA

<213> Artificial Synthesis

<400> 16

cggaccttta attcaaccc 19

<210> 17

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 17

ttcataccgt cccaccat 18

<210> 18

<211> 23

<212> DNA

<213> Artificial Synthesis

<400> 18

agcggataac aatttcacac agg 23

Claims (10)

1. The fusion protein of the recombinant forest encephalitis virus is characterized in that the fusion protein is forest encephalitis virus envelope protein E protein, and anchor protein PA3 and the forest encephalitis virus envelope protein E protein are connected in series by using a Linker to obtain the fusion protein.

2. The fusion protein of claim 1, wherein the amino acid sequence of Linker is shown in SEQ ID No. 4.

3. The fusion protein of claim 1, wherein the amino acid sequence of the anchor hook protein PA3 is set forth in SEQ ID No. 6.

4. The fusion protein of claim 1, wherein the forest encephalitis virus envelope protein E protein has an amino acid sequence shown in SEQ ID No. 2.

5. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is represented by SEQ ID No. 8.

6. A gene encoding the fusion protein of any one of claims 1-5.

7. The application of the forest encephalitis virus envelope protein E protein in preparation of a forest encephalitis virus vaccine is characterized in that the amino acid sequence of the forest encephalitis virus envelope protein E protein is shown as SEQ ID No. 2.

8. A recombinant tick-borne encephalitis virus bacteria-like particle vaccine, which is prepared by displaying the fusion protein of any claim 1-5 on the surface of GEM particle.

9. A method for preparing the fusion protein according to any one of claims 1 to 5, wherein the method comprises the following steps:

(1) connecting a gene encoding the anchor hook protein PA3 protein with a gene encoding the forest encephalitis virus envelope protein E protein through a gene encoding a Linker to obtain a recombinant gene;

(2) connecting the recombinant gene obtained in the step (1) with an expression vector pFastBac Dual to obtain a recombinant plasmid;

(3) transforming the recombinant plasmid obtained in the step (2) into a DH10Bac competent cell to obtain recombinant engineering bacteria, and extracting recombinant bacmid in the recombinant engineering bacteria;

(4) transfecting the recombinant bacmid obtained in the step (3) into Sf9 cells to obtain recombinant viruses, culturing the recombinant viruses to obtain cell suspension, wherein the fusion protein is in the cell suspension.

10. Use of the fusion protein of any one of claims 1-5 or the recombinant tick-borne encephalitis virus bacterial-like particle vaccine of claim 7 or 8 in preparation of a medicament for treating or preventing a disease infected with tick-borne encephalitis virus.

Images